Analog light measuring and photon counting in chemiluminescence measurements

ABSTRACT

A luminometer (400) includes a light detector (630) configured to sense photons (135). The luminometer (400) includes an analog circuit (915a) configured to provide an analog signal (965) based on the photons (135) emitted from assay reactions over a time period and a counter circuit (915b) configured to provide a photon count (970) based on the photons (135) emitted from the assay reactions over the time period. The luminometer (400) includes a luminometer controller (905) configured to, in response to an analog signal value of the analog signal (965) being greater than a predetermined value, determine and report a measurement value of the photons (135) emitted from the assay reactions over the time period based on the analog signal value of the analog signal (965) and a linear function (1010). Optionally, the linear function (1010) is derived from a relationship between the analog signal (965) and the photon count (970).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/560,636, filed Sep. 19, 2017, entitled SYSTEMFOR ANALOG LIGHT MEASURING AND PHOTON COUNTING IN CHEMILUMINESCENCEMEASUREMENTS, and to U.S. Provisional Patent Application No. 62/560,638,filed Sep. 19, 2017, entitled ANALOG LIGHT MEASURING AND PHOTON COUNTINGIN CHEMILUMINESCENCE MEASUREMENTS, the disclosures of which areincorporated herein in their entireties for all purposes. Thisapplication is related to PCT Patent Application, Attorney Docket No.087904-1063043-035110PC, filed on the same day herewith, entitled SYSTEMFOR ANALOG LIGHT MEASURING AND PHOTON COUNTING IN CHEMILUMINESCENCEMEASUREMENTS, the disclosure of which is incorporated herein in itsentirety for all purposes.

BACKGROUND

Many medical conditions can be diagnosed by sophisticated testing (i.e.,an assay) that includes combining sample fluids from a patient withreagents containing antibodies or antigens that are tailored to bondonly with an analyte in the sample fluid that the assay is meant tomeasure. The fluids and reagents are typically combined in a reactionvessel (e.g., a cuvette). At least one reagent can include a lightemitting enzyme (e.g., alkaline phosphatase) that directly or indirectlybonds with the analyte such that a greater amount of light emittedduring the testing indicates a greater amount of the analyte in thesample. During the testing process, a light detecting device, typicallya photomultiplier tube (“PMT”), is used to measure the amount of lightemitted (e.g., by counting apparent photons) by the light emittingenzymes. Electrical hardware connected to the PMT may provide an outputvalue related to the optical power of the emitted light by countingindividual photons that strike a photocathode of the PMT. The outputvalue may be transformed to a relative light unit (“RLU”) value.However, as a rate of the photons striking the photocathode increases,the probability of two or more photons striking the photocathodesimultaneously or nearly simultaneously increases. If two or morephotons strike the photocathode within a sufficiently small period oftime, these photons may only be counted as a single photon (i.e., anapparent single photon strike). The limit of the ability of a system todistinguish a pair of photons that strike nearly simultaneously is knownas pulse pair resolution. The previous electrical hardware cannotdistinguish a true single photon from a plurality of photons strikingtogether or nearly together when a time between strikes is below itspulse pair resolution and thus miscounts the photons and underreportsthe optical power received by the PMT. Previous systems have sufferedfrom various issues, including the undercounting of photons emitted athigh rates, that make their test results unreliable and/or insensitive.For example, as the optical power increases, the PMT becomes unable toaccurately measure the incoming light. The relationship between opticalpower of the light source and the electrical signal generated by theelectrical hardware connected to the PMT (which is used to calculate theRLU value of the light source) is or becomes non-linear. Additionally,non-linear output requires cumbersome analysis to provide an RLU value.Further, existing systems suffer from external light source photoncontamination, reaction vessel position inaccuracies, photoncontamination from adjacent reaction vessels, temperature variability,and so forth, all of which threaten to compromise the accuracy of thetest results. Accordingly, new and improved systems and methods areneeded.

BRIEF SUMMARY

Described herein are systems and methods for performing an assay using asystem that mitigates temperature disturbances, photon contaminationfrom external light sources, photon contamination from other reactionvessels, and reaction vessel position inaccuracies. Further, the systemsand methods described herein provide a substantial extension in thesignal linearity of the system output response over prior systems.Extended signal linearity improves the accuracy of test results thathave high output response values (relative light unit (“RLU”) values).Additionally, the systems and methods described herein provide signallinearity at low ranges through the crossover point to high ranges, suchthat there is no offset, which mitigates the need for manual analysis ofthe system results.

A system of one or more computers can be configured to performparticular operations or actions by virtue of having software, firmware,hardware, or a combination of them installed on the system that inoperation causes or cause the system to perform the actions. One or morecomputer programs can be configured to perform particular operations oractions by virtue of including instructions that, when executed by dataprocessing apparatus, cause the apparatus to perform the actions. Onegeneral aspect includes a luminometer system. The luminometer system mayinclude a light detector configured to sense photons emitted from assayreactions over a period of time. The luminometer system may also includean analog circuit configured to provide an analog signal based on thephotons emitted from the assay reactions over the period of time. Theluminometer system may also include a counter circuit configured toprovide a photon count based on the photons emitted from the assayreactions over the period of time. The luminometer system may alsoinclude a luminometer controller configured to, in response to an analogsignal value of the analog signal being greater than a predeterminedvalue, determine and report a measurement value of the photons emittedfrom the assay reactions over the period of time based on the analogsignal value of the analog signal and a linear function, where thelinear function is derived from a relationship established between theanalog signal and the photon count. Other embodiments of this aspectinclude corresponding computer systems, apparatus, and computer programsrecorded on one or more computer storage devices, each configured toperform the actions of the methods.

Implementations may include one or more of the following features. Theluminometer controller may be configured to, in response to the analogsignal value being less than the predetermined value, determine andreport the measurement value of the photons emitted from the assayreactions over the period of time based on the photon count. Optionally,the luminometer controller is further configured to perform a mastercalibration. In the master calibration, a photon measurement processormay set a gain of the light detector to operate in a plateau region. Thephoton measurement processor may calculate the predetermined value usingthe gain of the light detector. The photon measurement processor maycalculate a low voltage value of the light detector using the gain ofthe light detector. The photon measurement processor may also generatethe linear function using the predetermined value and the low voltagevalue.

Optionally, the luminometer system further includes a light emittingdiode arranged such that the light detector measures photons emitted bythe light emitting diode. Optionally, the luminometer controller isconfigured to apply a minimal voltage to the light emitting diode.Optionally, the luminometer controller is configured to increase theminimal voltage to a minimum readable voltage, the minimum readablevoltage being a minimum voltage applied to the light emitting diode tocause the light emitting diode to emit sufficient photons for the lightdetector to measure. The luminometer controller may also receive a ledphoton count and a led analog signal value each representing the photonsemitted from the light emitting diode when the minimum readable voltageis applied to the light emitting diode. The luminometer controller mayalso save the led analog signal value as the low voltage value.

The luminometer system may further include a light emitting diodearranged such that the light detector measures photons emitted from thelight emitting diode. The luminometer system may also include a lightsensor configured to sense light output of the light emitting diode andto generate a led analog output signal value representing photonsemitted from the light emitting diode. Optionally, the luminometercontroller may be configured to apply a minimum readable voltage to thelight emitting diode. The luminometer controller may also be configuredto increase the minimum readable voltage by a buffer voltage until theled analog output signal value representing the photons emitted from thelight emitting diode when the minimum readable voltage plus the buffervoltage is applied to the light emitting diode is greater than the lowvoltage value plus a buffer voltage value, where the buffer voltagevalue represents the photons emitted from the light emitting diode whenthe buffer voltage is applied to the light emitting diode. Theluminometer controller may also be configured to save the led analogoutput signal value as the predetermined value.

The luminometer system may further include a light emitting diodearranged such that the light detector measures photons emitted from thelight emitting diode. Optionally, the luminometer controller isconfigured to determine a minimum readable signal that generates the lowvoltage value from the light detector when the minimum readable signalis applied to the light emitting diode. Optionally, the luminometercontroller may also determine a high signal that generates thepredetermined value from the light detector when the minimum readablesignal plus a buffer voltage is applied to the light emitting diode. Theluminometer controller may also select a first voltage and a secondvoltage between the high voltage and the minimum readable voltage thatdivides the minimum readable voltage to the high voltage into four equalincrements. The luminometer controller may also receive a plurality ofcalibration analog signal values and a plurality of calibration photoncounts from the light detector based on applying each of the minimumreadable voltage, the high voltage, the first voltage and the secondvoltage to the light emitting diode. The luminometer system may alsoinclude perform a linear regression using the plurality of calibrationanalog signal values and the plurality of calibration photon counts togenerate the linear function. The luminometer system where theluminometer controller is configured to calculate the photon count byaccumulating pulse counts from the light detector over the predeterminedperiod of time using a boxcar averaging function. The system where theluminometer controller is configured to: convert and filter an outputsignal of the light detector to generate an analog output voltage. Theluminometer controller may be configured to collect the analog outputvoltage in increments over the predetermined period of time to generatea plurality of filtered voltage increments. The luminometer controllermay also average the filtered voltage increments to generate the analogvoltage value. The luminometer system may further include a lightemitting diode arranged such that the light detector measures photonsemitted from the light emitting diode. The luminometer system may alsoinclude where the luminometer controller is configured to perform a testcalibration cycle to correct a drift of the light detector, where thetest cycle calibration is based on the predetermined value and a lowvoltage value, and where the low voltage value represents photonsemitted from the light emitting diode when a minimum readable voltage isapplied to the light emitting diode. The luminometer system where theanalog circuit includes a first amplifier and where a signal to noiseratio at an input to the first amplifier is greater than 50 and lessthan 1,000,000. The luminometer system where the analog circuit includesa first amplifier and where a signal to noise ratio at an input to thefirst amplifier is greater than 500 and less than 4,000. The luminometersystem where a signal to noise ratio at an input to the analog circuitis greater than 100. The luminometer system where the analog signal isan analog voltage. The luminometer system where the light measurementvalue is a light power value. The luminometer system where the lightmeasurement value is a relative light unit (“RLU”) value. Theluminometer system where the light detector includes a photomultipliertube. Implementations of the described techniques may include hardware,a method or process, or computer software on a computer-accessiblemedium.

In one aspect, a luminometer system includes a photomultiplier tubeconfigured to sense photons emitted from an assay reaction over a periodof time. Optionally, the luminometer system further includes an analogcircuit configured to provide an assay analog voltage based on thephotons emitted from the assay over the period of time. Optionally, theanalog circuit may include a current sensing resistor coupled to convertcurrent from the photomultiplier tube to a voltage. The analog circuitmay also include an amplifier configured to amplify the voltage. Theanalog circuit may also include a dedicated electrical connectionbetween a terminal of the current sensing resistor and a terminal of theamplifier. The luminometer system may also include a counter circuitconfigured to provide an assay photon count based on the photons emittedfrom the assay over the period of time. Optionally, a luminometercontroller is configured to, in response to the assay analog voltagebeing greater than a predetermined value, calculating a relative lightunit (RLU) value of the photons emitted from the assay over the periodof time based on the assay analog voltage and an optimized linearfunction. Other embodiments of this aspect include correspondingcomputer systems, apparatus, and computer programs recorded on one ormore computer storage devices, each configured to perform the actions ofthe methods.

Implementations may include one or more of the following features. Theluminometer system where the current sensing resistor and the amplifierare disposed on a printed circuit board having a common ground plane.The luminometer system may also include the terminal of the currentsensing resistor and the terminal of the amplifier are connected to thecommon ground plane. Implementations of the described techniques mayinclude hardware, a method or process, or computer software on acomputer-accessible medium.

One general aspect includes a luminometer that includes a light detectorconfigured to receive photons emitted from a light source. The lightdetector may output a detector signal indicating a strength of the lightsource based on a number of photons received, where the detector signalincludes an analog component and a discreet component. The luminometermay also include a conversion circuit. The conversion circuit may beconfigured to receive the detector signal from the light detector. Theconversion circuit may also be configured to generate a digitized signalfrom the analog component of the detector signal, the digitized signalrepresenting the strength of the light source. Optionally, theconversion circuit may further be configured to generate a digitizedphoton count from the discreet component of the detector signal, thedigitized photon count representing an apparent number of photonsreceived by the light detector from the light source. The conversioncircuit may also be configured to output the digitized signal and thedigitized photon count. The luminometer may also include one or moreprocessors and a processor-readable memory having stored therein a setof instructions which, when executed by the one or more processors,cause the one or more processors to receive the digitized photon count.The instructions further cause the one or more processors to compare thedigitized photon count to a discreet-analog crossover value. Theinstructions further cause the one or more processors to output thedigitized photon count as a result from the luminometer when thedigitized photon count does not exceed the discreet-analog crossovervalue. When the digitized photon count exceeds the discreet-analogcrossover value, the instructions further cause the one or processors toapply a calibration function to the digitized signal to calculate anoutput value. The luminometer also includes output the output value asthe result from the luminometer. Other embodiments of this aspectinclude corresponding computer systems, apparatus, and computer programsrecorded on one or more computer storage devices, each configured toperform the actions of the methods.

BRIEF DESCRIPTION OF THE DRAWINGS

A further understanding of the nature and advantages of various examplesmay be realized by reference to the following figures.

FIG. 1 is a schematic diagram that illustrates an example assay processthat produces photons, according to an embodiment.

FIG. 2A is a graph that illustrates a representation of photon emissionoutput over a period of time from an example assay that produces lowphoton levels, according to an embodiment.

FIG. 2B is a graph that illustrates a representation of photon emissionoutput over a period of time from an example assay that produces highphoton levels, according to an embodiment.

FIG. 2C is a graph that illustrates a representation of photoelectronemissions over a period of time within a photomultiplier tube (“PMT”)receiving the example photon emission output illustrated at FIG. 2A.

FIG. 2D is a graph that illustrates a representation of photoelectronemissions over a period of time within a PMT receiving the examplephoton emission output illustrated at FIG. 2B.

FIG. 2E is a graph that represents an example signal output response ofthe PMT with the example photoelectron emission illustrated at FIG. 2C,the example signal output response having discreet pulses, according toan embodiment.

FIG. 2F is a graph that represents an example signal output response ofthe PMT with the example photoelectron emission illustrated at FIG. 2D,the example signal output response having overlapping pulses, accordingto an embodiment.

FIG. 2G is a graph that represents an example signal output response ofthe PMT with an example high-rate photoelectron emission, the examplesignal output response depicted with overlapped pulses, according to anembodiment.

FIG. 3 is an example graph of a prior art PMT output response inrelation to input optical power.

FIG. 4 is a perspective view illustrating an example luminometer forperforming an assay, according to an embodiment.

FIG. 5A is a front elevation view illustrating the example luminometerof FIG. 4 , according to an embodiment.

FIG. 5B is an enlarged portion of FIG. 5A, as called out at FIG. 5A.

FIG. 6A is a cross-sectional perspective view illustrating the exampleluminometer of FIG. 4 , according to an embodiment.

FIG. 6B is a cross-sectional side elevation view, as called out at FIG.5A, illustrating the example luminometer of FIG. 4 , according to anembodiment.

FIG. 7A is an enlarged portion of FIG. 6A, as called out at FIG. 6A.

FIG. 7B is an enlarged portion of FIG. 6B, as called out at FIG. 6B, butwith a reaction vessel removed.

FIG. 7C is an enlarged partial cross-sectional side view with a capclosed, illustrating the example luminometer of FIG. 4 , according to anembodiment.

FIG. 7D is an enlarged partial cross-sectional side view with the cap ofFIG. 7C opened, illustrating the example luminometer of FIG. 4 and aportion of a pick-and-place unit, according to an embodiment.

FIG. 8 is a schematic diagram that illustrates an example aperture viewof the luminometer of FIG. 4 , according to an embodiment.

FIG. 9A is a simplified block diagram that illustrates components of theluminometer of FIG. 4 , according to an embodiment.

FIG. 9B is a simplified circuit diagram of a conversion circuit,according to an embodiment.

FIG. 10A is an example graph of the luminometer output response inrelation to optical power input, according to an embodiment.

FIG. 10B is an enlarged portion of the example graph of FIG. 10A.

FIG. 11 is a flowchart depicting an example master calibration method ofa luminometer, according to an embodiment.

FIG. 12 is a flowchart depicting an example internal light sourcecalibration method of a luminometer, according to an embodiment.

FIG. 13 is a flowchart depicting an example testing method with extendedsignal linearity of a luminometer, according to an embodiment.

FIG. 14 is a flowchart depicting an example re-linearization method fora luminometer, according to an embodiment.

FIG. 15 is a schematic diagram that illustrates an example computersystem, according to an embodiment.

DETAILED DESCRIPTION

Embodiments described herein include systems and methods includingsystems using a photomultiplier tube (“PMT”) for performing an assay andsystems and methods with extended output signal linearity. The systemincludes components used to mitigate temperature disturbances, photoncontamination from external light sources, photon contamination fromother reaction vessels, and reaction vessel position inaccuracies thatimpact assay output measurements.

In the following description, for the purposes of explanation, specificdetails are set forth in order to provide a thorough understanding ofembodiments of the present disclosure. However, it will be apparent thatvarious embodiments may be practiced without these specific details. Thefigures and description are not intended to be restrictive.

Systems depicted in some of the figures may be provided in variousconfigurations. Optionally, the systems may be configured as adistributed system where one or more components of the system aredistributed across one or more networks in a cloud computing system. Allfeatures of the described systems are applicable to the describedmethods mutatis mutandis, and vice versa.

FIG. 1 illustrates an example assay 100. The example assay 100 may beperformed using the methods and/or systems of the present disclosure. Inaddition, other assays may also use the methods and/or systems accordingto the principles of the present disclosure. The assay 100 begins atstage 1. Reaction vessel 105 (e.g., cuvette) can be used for the assay100. A pipette 110 is used to place a first reagent 115 including ironparticles 120 coated with antibodies or antigens into the reactionvessel 105. The first reagent 115 can include antibodies or antigensthat are tailored to bond only with an analyte in a patient sample 165that the assay is meant to measure. In assay 100, the iron particles 120are coated with the antibodies or antigens.

At stage 2, the patient sample 165 is added to the reaction vessel 105with pipette 110. Pipette 110 may be cleaned, new, or have a new tip ateach stage. The patient sample 165 and the first reagent 115 are mixed.

At stage 3, the reaction vessel 105, containing the patient sample 165and the first reagent 115 (including the iron particles 120) (i.e.,mixture 170) are incubated with a heat source 125 to a predeterminedtemperature. During a binding process, the antibodies or antigens on theiron particles 120 of the first reagent 115 bind with the analyte ofinterest in the patient sample 165. The binding process can result inthe analyte of the patient sample 165 binding with the antigens orantibodies that are coated on the iron particles 120.

At stage 4, the reaction vessel 105 is moved near one or more magnets130, which attracts the iron particles 120 to one or more sides (e.g.,perimeter portions) of the reaction vessel 105. Pipette 110 is used towash the reaction vessel 105 with a washing agent 150. While washing,the magnet(s) 130 retain the iron particles 120 at the one or more sidesof the reaction vessel 105. The iron particles 120 and the bound analyteof the patient sample 165 remain in the reaction vessel 105 after thewashing is complete by virtue of the magnet(s) 130. Other components ofthe patient sample 165 may be absent from the reaction vessel 105 afterthe washing is complete, having been washed away by the washing agent150.

At stage 5, a second reagent 155, including alkaline phosphatase(“ALP”), can be placed in the reaction vessel 105 with the ironparticles 120 and the bound analyte of the patient sample 165 usingpipette 110. The second reagent 155 and the iron particles 120 can bemixed and incubated. The second reagent 155 can include an antibodyattached to the ALP that binds with the analyte of the patient sample165, still attached to the iron particles 120.

At stage 6 the magnet(s) 130 pull the iron particles 120 to one or moresides of the reaction vessel 105. The iron particles 120 now have thebound analyte of the patient sample 165 and the ALP of the secondreagent 155 bound to them. Unbound portions of the second reagent 155are rinsed away with additional washing agent 150 added with pipette 110to reaction vessel 105.

At stage 7, a substrate material 180 is added to the reaction vessel 105with pipette 110. The substrate material 180 is mixed and incubated. Thesubstrate material 180 reacts with the ALP enzyme and thereby produceslight 135 (i.e., photons).

At stage 8, the light 135, emitted by the reaction of the substratematerial 180 and the ALP attached to the iron particles 120, can beobserved 160 (i.e., by processes that are sensitive to light). Theobservation 160 can be performed by a light detector (e.g., a PMT). ThePMT can generate an output signal that can be processed to generate arelative light unit (“RLU”) value (i.e., an output response) indicatinga result of the assay 100. For example, a larger RLU value indicatesmore light, which indicates a larger amount of the analyte in thepatient sample 165 than a smaller RLU value indicates.

FIG. 2A illustrates an example low photon emission input graph 205depicting a low light level input to a PMT, according to an embodiment.Pulses 210 of low photon emission input graph 205 each indicate a singlephoton (e.g., emitted from the reaction of the ALP and the substratematerial 180 of assay 100, described above). Low photon emission inputgraph 205 therefore indicates a plurality of single photoelectronemissions. Because the pulses 210 are sufficiently spaced in time, eachof the single photon emissions is distinguishable and the individualphotons can be counted by the PMT, as described further with respect toFIGS. 2C and 2E.

FIG. 2B illustrates a high photon emission input graph 215, depicting ahigh light level input to a PMT, according to an embodiment. Pulses 220each indicate a single photon (e.g., emitted from the reaction of theALP and the substrate material 180 of assay 100, described above). Highphoton emission input graph 215 indicates multiple photoelectronemissions. In particular, individual photon emissions may becomeindistinguishable as they become closer in time, as described furtherwith respect to FIGS. 2D, 2F, and 2G. High photon emission input graph215 indicates the emission of photons at a higher light level thanindicated at graph 205.

FIG. 2C illustrates a low photoelectron emission graph 225 depictingindividual photoelectron emission that is measureable within a PMT usingthe photons indicated by pulses 210 of low photon emission input graph205 as the input (i.e., measured photons). Individual photons receivedby the PMT may each result in an emission of an individual photoelectronwithin the PMT. Each pulse 230 indicates a single photoelectron ejectedby a photon. Not every emitted photon is measurable and/or within areadable view of an aperture of the PMT. Accordingly, not every photonindicated by pulses 210 of low photon emission input graph 205 iscounted by the PMT.

FIG. 2D illustrates high photoelectron emission graph 235, whichrepresents the photoelectron emission that is measurable within the PMTusing the photons indicated by pulses 220 of high photon emission inputgraph 215 as the input (i.e., measured photons). Because not everyphoton is measurable and/or within the readable view of the aperture ofthe PMT, the photoelectron emission graph 235 does not depict aphotoelectron (indicated by pulses 240) for every photon indicated by apulse 220 in the high photon emission input graph 215.

FIG. 2E illustrates a discreet signal output response graph 245depicting the discrete pulse signal output of the PMT. Discreet signaloutput response graph 245 illustrates that the discrete pulses each havea time duration including a ramp-up portion and a ramp-down portion.Discreet signal output response graph 245 may be the signal outputresponse of the PMT with the example photoelectron emission illustratedin low photoelectron emission graph 225. The range of RLUs readable bythe PMT using a discrete pulse mode has an upper threshold, which variesfor each PMT. For example, the upper threshold may be up toapproximately 12 million to 14 million RLUs. Because the discrete pulsesof discreet signal output response graph 245 are separated in time, thepulses can be distinguished from each other and individual photoncounting is possible.

FIG. 2F illustrates overlapping pulse signal output response graph 250,which represents the discrete pulse signal output of the PMT when theinput optical power is too high to read all of the discreet pulsesignals. Overlapping pulse signal output response graph 250 illustratesthat the discrete pulses each have a time duration including a ramp-upportion and a ramp-down portion, and the ramp-up and ramp-down portionsoverlap the neighboring pulses. Overlapping pulse signal output responsegraph 250 may be the signal output response of the PMT with the examplephotoelectron emission illustrated in high photoelectron emission graph235. Because the discrete pulses are not cleanly separated in time, theycannot always be distinguished from each other and pulse counting is notpossible without losing a significant number of pulses in the count.

FIG. 2G illustrates overlapped signal output response graph 255, whichrepresents the pulse overlapped signal output of the PMT with theexample photoelectron emission illustrated in a high photoelectronemission graph. The pulse overlapped signal output can be measured by anelectrical current (i.e., amperage) output of the PMT, and an RLU valuecan be generated according to embodiments described herein.

As shown in FIGS. 2A-2G, at lower input light levels (i.e., light levelsgenerating an output response below the discreet/analog crossover), avast majority of the discrete pulses of the photoelectrons can bedistinguished, and an output response RLU value can be accuratelydetermined based on individual photon counting. At higher input lightlevels (i.e., light levels generating an output response above thediscreet/analog crossover), many of the discrete pulses of thephotoelectrons cannot be distinguished, and an output response RLU valuecannot be accurately determined based on individual photon counting. Athigher light levels, the analog current output of the PMT can bemeasured, can be converted to a voltage that is measured, and/orotherwise processed as a signal to determine the RLU output, asdescribed further herein.

FIG. 3 illustrates an example graph 300 of an output response of a priorart luminometer with a typical PMT. The example graph 300 depicts theRLU output response of the luminometer with respect to input opticalpower. A PMT outputs a signal having a discreet component. The discreetcomponent provides a signal representing a discreet photon count. As thephoton rate increases, an increasing number of counts are lost becauseof pulse overlap, as described above. Luminometers include electricalhardware and circuitry to obtain the PMT output signal and convert theoutput signal into an RLU output response. In the prior art luminometer,illustrated by the example graph 300, the relationship between the RLUoutput response of the luminometer and the input optical power isnon-linear. As seen by graph 300, with the prior art luminometer, as theoptical power increases, the RLU output response of the luminometerbecomes non-linear as more photon counts are missed, which results incurve 305, which is non-linear. Photons cannot be individually countedat higher light levels (i.e., higher optical power) without missing asubstantial number of photon counts because the PMT is unable to detectindividual photons whose pulses substantially overlap in time. This isshown by high photon emission input graph 215, high photoelectronemission graph 235, overlapping pulse signal output response graph 250,and overlapped signal output response graph 255. Further, each PMT iscalibrated in one of multiple ways, each of which may impact the RLUoutput of the PMT at higher light levels. For example, graph 300 is anexample output response from an Access 2, DxI 600, or DxI800.

FIG. 4 illustrates a perspective view of a luminometer 400 forperforming an assay 100, according to an embodiment. Luminometer 400mitigates increasing inaccuracies at higher input optical power as wellas other inaccuracies including photon contamination, reaction vesselposition inaccuracies, and thermal variances. FIG. 4 provides anexternal view of luminometer 400.

Luminometer 400 includes a chassis 405. In certain embodiments, thechassis 405 can be any material that promotes thermal and electricalconductance and thermal consistency, such as, for example, aluminum. Thechassis 405 includes a chassis portion 535 of a labyrinth seal 530depicted in more detail in FIGS. 5B, 7A, 7B, and 7C. At the center ofthe chassis portion 535 of the labyrinth seal 530 is a chamber opening430 providing access to a reaction vessel chamber 610 (see FIG. 7B).

Luminometer 400 includes a cap 415. The cap 415 can be the same materialas the chassis 405, such as, for example aluminum. As a more specificexample, 6061-T6 aluminum can be used for aluminum portions of theluminometer 400 including the cap 415 and the chassis 405. The cap 415can form a non-contacting photon seal when closed over the chassisportion 535 of the labyrinth seal 530 of the chassis 405 and therebyform a dark chamber 545 (see FIG. 7C). Stated differently, the cap 415can be placed over the chassis portion 535 of the labyrinth seal 530 ofthe chassis 405 to restrict external light (light emitted from externalsources) from entering the reaction vessel chamber 610. Optionally, acap portion 540 of the labyrinth seal 530 (shown in more detail in FIGS.5B, 7A, and 7C), which is included on the cap 415, does not makephysical contact with the chassis portion 535 of the labyrinth seal 530.Optionally, the cap 415 can be considered closed (i.e., in a closedstate or closed configuration 555) when external light entering thereaction vessel chamber 610 is restricted sufficiently that a PMT 630does not measure external light (i.e., light introduced within thereaction vessel chamber 610 by a source outside of chassis 405 and/orcalibration unit 460). The labyrinth seal 530 may be made of and/orcoated with light absorbing material. For example, the cap portion 540of the labyrinth seal 530 and/or the chassis portion 535 of thelabyrinth seal 530 may absorb light. Thus, even though the labyrinthseal 530 may be non-contacting, light may not reflect within clearancesof the labyrinth seal 530 to reach the reaction vessel chamber 610 whenthe labyrinth seal 530 is in the closed configuration 555. When a bottom685 of the cap 415 is parallel to (even though optionally notcontacting) a top 680 of the chassis 405 and the chassis portion 535 ofthe labyrinth seal 530, and the cap portion 540 and the chassis portion535 of the labyrinth seal 530 engage due to the intermeshing walls, thecap 415 is in a closed configuration 555. The cap 415 can be lifted andlowered by a cap arm 420, as described in further detail below. Once anopening angle 655 (see FIG. 6B) between the bottom 685 of the cap 415and the top 680 of the chassis 405 reaches approximately 7 degrees ormore, the cap 415 enters an open configuration 560 (i.e., the cap 415 isopen or in an open state). As depicted in FIG. 4 , the cap 415 is in anopen configuration 560. Upon the opening angle 655 of the cap 415reaching a sufficient angle (e.g., 90 degrees), a chamber opening 430 tothe reaction vessel chamber 610 is accessible by, for example, apick-and-place unit 790 (see FIG. 7D) which may deliver a reactionvessel 105 to the reaction vessel chamber 610 and/or retrieve thereaction vessel 105 from the reaction vessel chamber 610. An examplegripper assembly that is suitable for use with the pick-and-place unit790 is illustrated at U.S. Pat. No. 7,128,874 B2, incorporated herein byreference in its entirety.

The cap arm 420 can cause the cap 415 to open and close. The cap arm 420can be controlled (i.e., actuated) by an actuator 425 (e.g., a motor425). As depicted, the actuator 425 is a stepper motor 425.

Luminometer 400 can include a computer system 1500 that is configured asa luminometer controller 905 and a luminometer computer systemcompartment 435. The luminometer computer system compartment 435 canhouse the computer system portion of the luminometer 400 that isdescribed in more detail with respect to FIG. 9A.

The luminometer 400 can include a sensor (not shown) that can detectwhether the cap 415 is closed or open and/or is entering a closed stateor entering an open state. The cap sensor can send a signal to theluminometer controller 905 (within luminometer computer systemcompartment 435) that can register the sensor signal and, when the cap415 is entering an open state or is open (i.e., is in an openconfiguration 560), send a signal to close a shutter 660 to protect thePMT 630 in the luminometer 400 from damage due to light entering thereaction vessel chamber 610 through the chamber opening 430. When thecap 415 is entering a closed state 555 or is closed, the sensor can senda signal to the luminometer controller 905. The luminometer controller905 can register the signal and open the shutter 660 to allow the PMT630 to view light sources within the light passage 640 and the reactionvessel chamber 610 (i.e., the dark chamber 545). The shutter 660 can bean electronic shutter 660 such that it automatically opens and closesupon receiving the signal from the sensor. The shutter 660 can be asolid state electronic shutter 660 (i.e., no moving parts).

Luminometer 400 can include a stand 440 for supporting the chassis 405,the computer system compartment 435, and the other components ofluminometer 400. A thermal barrier 445 can separate the chassis 405 fromthe stand 440. As depicted, the thermal barrier 445 also serves as aposition adjustment for the luminometer 400 (e.g., to align the reactionvessel chamber 610 with the pick-and-place unit 790). In otherembodiments, the thermal barrier 445 may be separate from thepick-and-place unit 790. Stated differently, a thermal barrier 445 canbe placed on the stand 440 and the chassis 405 can be placed on thethermal barrier 445. The thermal barrier 445 can be plastic to mitigatethermal transfer between the chassis 405 and the stand 440 as well asbetween the chassis 405 and the luminometer computer system compartment435.

Luminometer 400 can include a PMT cover 450 that can be any materialthat promotes thermal and electrical conductance and thermalconsistency, such as, for example, aluminum (e.g., 6061-T6 aluminum).The PMT cover 450, assembled to a housing 410 of the chassis 405, canform an enclosure 565 (see FIG. 7C) that houses the PMT 630. Theenclosure 565 can mitigate thermal transfer between the chassis 405 andthe external environment. A heating element 455 (e.g., a heatingblanket) can surround the chassis 405 and be controlled by a thermistor(not shown) within a thermistor passage (also called a thermistorchannel) in the chassis 405. The thermistor can measure a temperature ofthe chassis 405 and/or the enclosure 565. In other embodiments, anytemperature sensor may be used in place of or together with thethermistor. The thermistor can transmit a temperature signal that can besent to the luminometer controller 905 of the luminometer 400 which canmaintain a constant temperature of the chassis 405 and/or the enclosure565 by turning the heating element 455 on when the temperature falls andturning the heating element 455 off when the temperature reaches apredetermined set point. A constant temperature within the chassis 405can mitigate thermal variance discrepancies (i.e., inaccuracies) withthe assay results. An optimal constant temperature can be, for example,37 degrees Celsius. Luminometer 400 can further include heating elementcover 480. Heating element cover 480 can help discourage thermaltransfer to the external environment from heating element 455. The PMTcover 450 can be coupled to the chassis 405 with a conductive gasket 485(see FIGS. 6A and 7C). The conductive gasket 485 can promoteelectromagnetic interference shielding within the chassis 405 and/or theenclosure 565 as well as promoting thermal consistency in luminometer400. The conductive gasket 485 can provide thermal conductivity as wellas electrical conductivity between the chassis 405 and the PMT cover450. The chassis 405 and the PMT cover 450 can create a thermal cavitydefining an enclosed volume that houses the PMT 630, the reaction vesselchamber 610, and the light passage 640. The cavity and the dark chamber545 can thus be free of external light, at a constant temperature, andshielded from electromagnetic interference.

Luminometer 400 can also include a calibration unit 460 (e.g., anon-board calibration unit 460). The calibration unit 460 can be used tocalibrate the PMT 630, as described with respect to FIGS. 9A, 11, and 14. The calibration unit 460 can have a receptacle 475 for providing powerinput to the calibration unit 460 and signal output from the calibrationunit 460. The signal output of the calibration unit 460 can betransmitted to the luminometer controller 905 in luminometer computersystem compartment 435.

Luminometer 400 can also include a PMT voltage input socket 470 a whichis coupled to a PMT voltage input socket 470 b and provides power inputto the PMT 630. Luminometer 400 can also include a luminometer outputsignal socket 465 a which is coupled to a luminometer output signalsocket 465 b. The PMT voltage input socket 470 a and the PMT voltageinput socket 470 b can be coupled with a cable that allows a voltagepower to be sent from the luminometer computer system compartment 435 tothe PMT 630 for powering the PMT 630. The luminometer output signalsocket 465 a and the luminometer output signal socket 465 b can becoupled with a cable that allows the output signal of the PMT 630 to besent from the PMT 630 to the luminometer computer system in theluminometer computer system compartment 435. While PMT voltage inputsocket 470 a and luminometer output signal socket 465 a are shown placedhorizontally next to each other at FIGS. 4, 5A, and 6A, otherconfigurations and locations for the sockets 470 a and 465 a are withinthe scope of this disclosure (e.g., at FIGS. 6B and 7C).

FIG. 5A illustrates a front elevation view 500 of the luminometer 400,according to an embodiment. The front elevation view 500 depicts the cap415, the chassis 405, the luminometer computer system compartment 435,the stand 440, the motor 425, the heating element cover 480, and thethermal barrier 445. Also shown is the PMT voltage input socket 470 a,the PMT voltage input socket 470 b, the luminometer output signal socket465 a, and the luminometer output signal socket 465 b. The side view 500also shows both the cap portion 540 of the labyrinth seal 530 and thechassis portion 535 of the labyrinth seal 530. As shown by the dashedcircle, an enlarged portion of the front elevation view of the labyrinthseal 530 is provided in FIG. 5B.

FIG. 5B illustrates an enlarged view 550 of the labyrinth seal 530formed by the cap portion 540 of the labyrinth seal 530 and the chassisportion 535 of the labyrinth seal 530. The cap portion 540 of thelabyrinth seal 530 is included with the cap 415. Between a cap outerconcentric ring 505 (i.e., a wall) and a cap inner concentric ring 510(i.e., a wall) is a cap trough 515 (i.e., a wall receiver). The capouter concentric ring 505, cap inner concentric ring 510, and trough 515form the cap portion 540 of the labyrinth seal 530. The chassis portion535 of the labyrinth seal 530 includes a chassis outer concentric ring520 (i.e., a wall), a chassis inner concentric ring 525 (i.e., a wall),a chassis inner trough 725 (i.e., a wall receiver and a chassis outertrough 730 (i.e., a wall receiver). Chassis inner concentric ring 525 isthe innermost (i.e., centermost) ring and may be taller than the chassisouter concentric ring 520. As depicted, the chassis inner concentricring 525 is a circular wall and having a larger height can facilitateexclusion of light in the reaction vessel chamber 610 even when thebottom 685 of the cap 415 is not parallel with the top 680 of thechassis 405. The external diameter (i.e., the diameter of the outermostedge) of the chassis inner concentric ring 525 is larger than thediameter of the chamber opening 430 allowing access to the reactionvessel chamber 610. Between the chassis inner concentric ring 525 andchassis outer concentric ring 520, there is a chassis inner trough 725that has an external diameter larger than the external diameter of thechassis inner concentric ring 525. Outside the chassis outer concentricring 520 (which has an external diameter larger than the externaldiameter of the chassis inner trough 725), there is a chassis outertrough 730 that has an external diameter larger than the externaldiameter of the chassis outer concentric ring 520. The cap portion 540of the labyrinth seal 530 can engage with (i.e., mesh with) the chassisportion 535 of the labyrinth seal 530 when in a closed configuration 555(see FIG. 7C). When closed, the cap trough 515 can fit over the chassisouter concentric ring 520 of the chassis portion 535 of the labyrinthseal 530. The cap trough 515 can have an external diameter that islarger than the external diameter of the chassis outer concentric ring520. The cap inner concentric ring 510 can fit within chassis innertrough 725 and the cap outer concentric ring 505 can fit within chassisouter trough 730.

FIG. 6A illustrates a cross-sectional perspective view 600 of theluminometer 400 for performing the assay 100, according to anembodiment. The cross-sectional perspective view 600 provides a cut-awayview of luminometer 400 as shown by the cross-sectional cutting line inFIG. 5A. The cut portions of the cross-sectional perspective view 600are shown by cross-hatching. The cross-sectional perspective view 600illustrates the cap 415, the chassis 405, the luminometer computersystem compartment 435, the PMT cover 450, the stand 440, the motor 425,the thermal barrier 445, and the calibration unit 460. Also shown is theluminometer output signal socket 465 a and the luminometer output signalsocket 465 b. As indicated by the dashed circle, FIG. 7A provides anenlarged view of a portion of the cross-sectional perspective view 600of the luminometer 400.

The cross-sectional perspective view 600 provides a view of the chamberopening 430, which provides access to reaction vessel chamber 610.Reaction vessel 105 is shown seated within reaction vessel chamber 610.Light passage 640 intersects with reaction vessel chamber 610 near thebottom of the reaction vessel chamber 610.

The cross-sectional perspective view 600 further provides a view of thePMT 630. The PMT 630 can be a photomultiplier tube or any other suitablelight detecting device or light detector. The PMT 630 can include asensing element (not shown in detail) that detects light from lightpassage 640 and/or the reaction vessel chamber 610. The PMT 630 isadjacent an aperture 635 that is aligned with the light passage 640 andpast an intersection of the light passage 640 and the reaction vesselchamber 610. The aperture 635 allows light to enter the PMT 630 and thesensing element to receive the light. The reaction vessel chamber 610intersects with the light passage 640 such that when the reaction vessel105 is placed in the reaction vessel chamber 610, the substance orsample within the reaction vessel 105 can emit photons viewable in thelight passage 640 and to the aperture 635. The aperture 635 can belimited in size, for example to 8.5 centimeters in diameter, to limitthe view of a meniscus 815 within the reaction vessel 105 as discussedin more detail with respect to FIG. 8 . On the other end of the lightpassage 640, the calibration unit aperture 645 can align with the lightpassage 640. The calibration unit 460 can include a light emitting diode(“LED”) 620 and a photodiode 625. The LED 620 and photodiode 625 canprovide a regulated internal light source used to calibrate PMT 630. Thereaction vessel 105 is not needed in the luminometer 400, for exampleduring calibration. While the luminometer 400 is described as includingthe reaction vessel 105, this is an optional component of the systemthat may not necessarily be part of the luminometer 400.

FIG. 6B illustrates a cross-sectional side elevation view 650 ofluminometer 400 for performing the assay 100. The cross-sectional sideelevation view 650 provides a cut-away view as thought the luminometer400 were cut by the cross-sectional cutting line in FIG. 5A. The cutportions of the cross-sectional side view 650 are shown bycross-hatching. The cross-sectional side view 650 depicts the cap 415,the chassis 405, the cap arm 420, the motor 425, the luminometercomputer system compartment 435, the PMT cover 450, the stand 440, themotor 425, the thermal barrier 445, and the calibration unit 460. Alsoshown are the luminometer output signal socket 465 a and the luminometeroutput signal socket 465 b. As indicated by the dashed circle, FIG. 7Bprovides an enlarged portion of the cross-sectional side view 650 of theluminometer 400.

Cross-sectional side elevation view 650 indicates that opening angle 655is the angle between a bottom 685 of the cap 415 and the top 680 of thechassis 405. As the cap 415 moves toward the closed configuration 555,the opening angle 655 becomes smaller. Once the opening angle 655reaches approximately seven degrees or less, the cap 415 is in a closedconfiguration. When the cap 415 is in a closed configuration 555, a darkchamber 545 is formed within reaction vessel chamber 610. The darkchamber 545 is formed when no external light (i.e., light from lightsources external to chassis 405) can enter the dark chamber 545. Thedark chamber 545 includes the light passage 640 and the reaction vesselchamber 610.

The cross-sectional side elevation view 650 further provides anotherview of the PMT 630. As shown in this cross-sectional side elevationview 650, the light passage 640 intersects with reaction vessel chamber610 on one end. On the other end, the light passage 640 is coupled tothe calibration unit 460. The calibration unit 460 can include the LED620 and the photodiode 625. The calibration unit aperture 645 allowslight from the calibration unit 460 to pass into the light passage 640.

FIG. 7A illustrates an enlarged partial perspective view 700 of thecross-sectional perspective view 600 of the luminometer 400 forperforming the assay 100, according to an embodiment. The enlargedperspective view 700 is identified by the dashed circle incross-sectional perspective view 600 of FIG. 6A. Shown in enlargedperspective view 700 are cap 415 with cap outer concentric ring 505, capinner concentric ring 510, and cap trough 515 to form the cap portion540 of the labyrinth seal 530. Further shown in enlarged perspectiveview 700 is the chassis portion 535 of the labyrinth seal 530 formed bychassis outer concentric ring 520, chassis inner concentric ring 525,chassis outer trough 730, and chassis inner trough 725. Enlargedperspective view 700 further depicts reaction vessel 105, reactionvessel chamber 610, PMT 630, aperture 635, calibration unit aperture645, and light passage 640.

Enlarged perspective view 700 shows additional detail of calibrationunit 460, including filter 715, which may cover calibration unitaperture 645. Filter 715 can filter the light emission from LED 620 intolight passage 640.

Enlarged perspective view 700 shows additional detail of reaction vesselchamber 610 and reaction vessel 105. A kinematic spherical joint 705 isdepicted. The kinematic spherical joint 705 can include a first portion745 (see FIG. 7B) that is a partial sphere, a cone, a partial cone, orother functional geometry that is located at, adjacent, or toward thebottom of the reaction vessel chamber 610. The reaction vessel 105 caninclude a second portion 735 of the kinematic spherical joint 705 thatmay be a convex spherical nose that mates to the first portion 745 ofthe kinematic spherical joint 705. As depicted, the second portion 735includes a partial spherical nose. The kinematic spherical joint 705 canlimit three degrees of freedom of the reaction vessel 105 (i.e., threemutually orthogonal directions of a point centered at the second portion735). Additionally, the reaction vessel 105 can have a first portion 710of a kinematic cylindrical joint 720 that is a fin 710 that fits withina second portion 740 of the kinematic cylindrical joint 720. As depictedin FIG. 7B, the second portion 740 is a cylindrical feature of thereaction vessel chamber 610 and defines an axis 675. In otherembodiments, other geometry may be used. The kinematic cylindrical joint720 is formed by the second portion 740 in conjunction with the fin 710and can limit two additional degrees of freedom of the reaction vessel105 (i.e., two mutually orthogonal rotational directions about the pointcentered at the second portion 735). The only remaining degree offreedom is vertical axial rotation about an axis 775 of symmetry of thereaction vessel 105. For the purposes of the assay 100 in luminometer400, vertical axial rotation will not impact the assay 100, andtherefore need not be limited. The second portion 740 of the reactionvessel chamber 610 can be sized such that the clearance between an outerdiameter of the fin 710 of the reaction vessel 105 and the innerdiameter of the second portion 740 of the reaction vessel chamber 610 isvery small, for example, less than 0.001 inch. The fin 710 may be spacedless than a fin thickness from (i.e., below) the tapered portion 765 ofthe reaction vessel chamber 610. The outer diameter of the fin 710 canbe, for example, 10.182 millimeters+/−0.16 millimeter. The outerdiameter of the fin 710 can interface with the second portion 740 of thekinematic cylindrical joint 720 that can be, for example, 10.414millimeters+/−0.12 millimeter in inner diameter. The sizing can provideclearance for the fin 710 to facilitate removal of the reaction vessel105 from the reaction vessel chamber 610. A clearance groove 760 mayfacilitate the insertion of a collet 795 of the pick-and-place 790 unitinto the chamber opening 430. A tapered portion 755 may guide the collet795 as it positions the reaction vessel 105 into the reaction vesselchamber 610. The tapered portion 765 may guide the outer diameter of thefin 710 as the reaction vessel 105 is positioned into the reactionvessel chamber 610. The tapered portions 755 and 765 prevent bindingwhen the reaction vessel 105 (and the collet 795) is removed from thereaction vessel chamber 610.

FIG. 7B illustrates an enlarged partial side view 750 of thecross-sectional side elevation view 650 of the luminometer 400 forperforming the assay, according to an embodiment. The enlarged side view750 is shown by the dashed circle in cross-sectional side view 650 ofFIG. 6B. This enlarged view 750 provides a more detailed view of thearea around reaction vessel chamber 610.

The chassis portion 535 of the labyrinth seal 530 is shown, with chassisinner trough 725 being formed between chassis inner concentric ring 525and chassis outer concentric ring 520. Chassis outer trough 730 isformed outside of chassis outer concentric ring 525. Chassis innerconcentric ring 525 surrounds chamber opening 430. Alternatively, onecan say that chassis inner trough 720 and chamber opening 430 formchassis inner concentric ring 525, and one can say that chassis outertrough 730 and chassis inner trough 725 form chassis outer concentricring 520.

As previously discussed, the reaction vessel 105 can have a firstportion 710 of a kinematic cylindrical joint 720 that is a fin 710 thatfits within a second portion 740 of the kinematic cylindrical joint 720that is a cylindrical feature of the reaction vessel chamber 610. Theclearance groove 760 has above it the tapered portion 755 which canguide the collet 795 and/or the reaction vessel 105 into the reactionvessel chamber 610. Below the clearance groove 760 may be the taperedportion 765 to guide or further guide the reaction vessel 105 into thereaction vessel chamber 610. The chamber opening 430 above the taperedportion 755 provides external access to the reaction vessel chamber 610.When the reaction vessel 105 is seated within the reaction vesselchamber 610, the bottom (i.e., the nose 735) of the reaction vessel 105engages with the first portion 745 and thereby forms the kinematicspherical joint 705.

The light from the light passage 640, after passing through the reactionvessel chamber 610 and reaction vessel 105, if present, enters the PMT630 through aperture 635. Photons emitted from a sample in reactionvessel 105 also enter PMT 630 through aperture 635.

FIG. 7C illustrates an enlarged partial side view of the luminometer 400with the cap 415 closed so that the luminometer 400 is in a closedconfiguration 555. The labyrinth seal 530 is engaged with the concentricrings 520, 525 of the chassis portion 535 of the labyrinth seal 530intermeshed with the concentric rings 505, 510 of the cap portion 540 ofthe labyrinth seal 530.

Luminometer 400 may be used to perform assays. In use, cap 415 can beopened, which can trigger a sensor to send a signal to the luminometercomputer system that can cause a shutter 660 to close to protect the PMT630 from damage due to overexposure to light. A reaction vessel 105 canbe positioned within the reaction vessel chamber 610. The reactionvessel 105 can contain a light emitting sample. When placed in thereaction vessel chamber 610, the spherical nose 735 of the reactionvessel 105 can secure the reaction vessel 105 via the kinematicspherical joint 705, and the fin 710 can secure the reaction vessel 105via the kinematic cylindrical joint 720. (As the thickness of the fin710 is small in relation to the outer diameter of the fin 710, the joint720 primarily constrains a point at the center of the fin 710 to theaxis 765 of the cylinder 740 of the reaction vessel chamber 610.) Whileno specimen is depicted in reaction vessel 105 within luminometer 400, aspecimen or sample, such as the patient sample 165 as described withrespect to FIG. 1 can be in the reaction vessel 105.

After the reaction vessel 105 is secured, the cap 415 can close toengage the cap portion 540 of the labyrinth seal 530 with the chassisportion 535 of the labyrinth seal 530 to form a dark chamber 545 (i.e.,to exclude external light from the reaction vessel chamber 610 and fromthe light passage 640). Once the cap 415 is closed, the sensor can senda signal to the luminometer computer system (e.g., the luminometercontroller 905) that can cause the shutter 660 to open, exposing theaperture 635 and thereby PMT 630 to the light passage 640 and/or thereaction vessel chamber 610. The PMT 630 can detect the photons emittingfrom the sample in the reaction vessel 105 through the aperture 635. Thephoton emission can be processed by the luminometer 400 as described inmore detail with respect to FIGS. 9A-14 .

After a threshold period of time, for example 1 second, the assayreading can be completed by the PMT 630. Multiple assay reads may beperformed on the same sample sequentially. A median value of the readsmay be reported as the result. The cap 415 can open, the reaction vessel105 can be removed from the reaction vessel chamber 610 (e.g., by thecollet 795 of the pick-and-place unit 790), and the cap 415 can close.Once closed with no reaction vessel 105 in the reaction vessel chamber610, test cycle calibration can be run using the calibration unit 460.Referring to FIG. 6A, the LED 620 can emit a known photon or light powervalue based on an input voltage to the LED 620. The photodiode 625 canmeasure the photon emission to confirm the LED 620 is functioningproperly and/or to provide feedback control. For example, the photodiode625 can send a signal indicating the LED output value to the luminometercomputer system (e.g., the luminometer controller 905), which canconfirm the value. Output of the photodiode 625 may be used as feedbackto control the output of the LED 620. The light from the LED 620 canshine off of the interior of the calibration unit housing (which can be,for example, anodized aluminum), and emit from the calibration unit 460through calibration unit aperture 645. Referring back to FIG. 7A, filter715 can filter the light emission from LED 620, and the filtered lightcan travel through light passage 640 to aperture 635. With no reactionvessel 105 present, the light can also travel up the reaction vesselchamber 610. The light that reaches aperture 635 can be measured by PMT630. The measured light can be processed to calibrate the PMT 630 asdescribed in further detail herein. The processes of performing an assay100 and test cycle calibration can repeat. Optionally, the test cyclecalibration can be executed between each assay. Optionally, the testcycle calibration can be executed less frequently.

FIG. 8 illustrates an example aperture view 800 of a luminometer,according to an embodiment. As discussed with respect to FIG. 7A, theaperture 635 can be sized sufficiently to mitigate the inaccuracies ofmeasuring a sample that includes a meniscus. FIG. 8 illustrates aportion of a reaction vessel 105 containing a sample 810 with a meniscus815. The unshielded view 820 is exposed to the inaccuracies associatedwith the meniscus 815. A meniscus 815 can have bubbles and/or otherunevenness that can cause the photon measurement within the luminometer400 to be inaccurate. The shielded aperture view 825 can obtain a moreaccurate reading because it is shielded from the meniscus 815. Theshielded aperture view 825 may be further reduced in size to wholly viewonly a portion of the reaction vessel 105 containing the sample 810. Insome embodiments, shielded aperture view 825 can be implemented via abracket 665 used to couple the PMT 630 to the chassis 405. The shieldedaperture view 825 can be shielded by the bracket 665 (i.e., a lightdetector bracket) that couples the PMT 630 to the chassis 405. Thebracket 665 can form a portion or a whole of the aperture 635 to createthe shielded aperture view 825.

FIG. 9A illustrates a simplified block diagram of a luminometerelectrical system 900, according to an embodiment. The luminometerelectrical system 900 can include the PMT 630, a conversion circuit 915,a luminometer controller 905, and a display device 920. While specificcomponents and modules are depicted, functionality of one or moremodules can be incorporated into a single module. Similarly,functionality of a single module can be spread amongst multiple moduleswithout affecting the scope of this disclosure. Luminometer controller905 may include additional modules and/or functionality than describedbelow or depicted in FIG. 9A. For example, luminometer controller 905may receive signals from sensors on luminometer 400 that luminometercontroller 905 processes and that may cause other actions. As anexample, luminometer controller 905 may receive a signal from a sensorindicating that the cap 415 is entering an open configuration.Luminometer controller 905 may, in response, send a signal to shutter660 to close so that external light cannot enter PMT 630 to avoiddamaging PMT 630.

The PMT 630 can receive a light emission through an aperture, such asluminometer aperture 635. The aperture 635 can render shielded apertureview 825. The PMT 630 outputs PMT output signal 975 based on thereceived light emission. The PMT output signal 975 can include twocomponents including analog current. The PMT output signal 975 can bereceived by the conversion circuit 915. The conversion circuit 915 isshown in more detail in FIG. 9B.

FIG. 9B illustrates the conversion circuit 915, which includes front endcircuitry. The front end circuitry may include sense resistor R_(S),that converts analog current in PMT output signal 975 into analogvoltage 985. The conversion circuit 915 may be configured to receive andcollect the PMT output signal 975 in increments over a period of time.For example, the PMT output signal 975 may be collected in 20millisecond increments for a second to collect 50 increments. The valuescollected in each increment may then be averaged to generate a singlevalue. The conversion circuit 915 includes an analog circuit portion 915a and a photon counter portion 915 b. The analog circuit portion 915 ais configured to convert the analog voltage 985 into an assay analogvoltage 980 (i.e., an analog voltage). In other embodiments, an analogcircuit portion is configured to transfer the analog current from PMToutput signal 975 as an assay analog current (i.e., an analog current).Such an analog voltage and such an analog current may be collectivelyreferred to as an analog signal. The analog circuit portion 915 aincludes a first stage amplifier, OA₁ (e.g., an operational amplifier,op-amp, etc.) and may include signal conditioner 913 including, forexample, additional amplifiers and/or signal filters. The assay analogvoltage 980 is digitized by analog to digital (A/D) converter 914 andthe digitized assay analog voltage 965 is transmitted to the interface925 of luminometer controller 905. The analog circuit portion 915 aincludes a dedicated electrical connection T₁ between a terminal N₁ ofthe current sensing resistor R_(S) and a terminal N₂ of the first stageamplifier OA₁. If the analog circuit portion 915 a is disposed on aprinted circuit board, the terminal N₁ of the current sensing resistorR_(S) and/or the terminal N₂ of the first stage amplifier OA₁ may alsobe connected to the ground plane of the conversion circuit 915. Thededicated electrical connection T₁, being connected directly between theresistor terminal N₁ and the amplifier terminal N₂, carries the returnsignal for the analog voltage 985, and does not substantially carryother signals which may be present on the ground plane. The presence ofsignals other than the return signal between the terminals N₁, N₂ of theresistor R_(S) and the first stage amplifier OA₁ reduces the signal tonoise ratio at the first stage amplifier terminal N₂. The use of thededicated electrical connection T₁ can provide a signal to noise ratiobetween approximately 10 to 1,000,000, for example, approximately 100.In certain embodiments, the use of the dedicated electrical connectionT₁ can provide a signal to noise ratio between approximately 500 to4,000. For example, in some scenarios, the use of the dedicatedelectrical connection T₁ can increase the signal to noise ratio at theterminal N₂ of the first stage amplifier OA₁ by at least a factor ofapproximately 10 or even a factor of approximately 100 when compared toan identical circuit that does not include the dedicated electricalconnection. As shown in FIG. 9B, the conversion circuit 915 furtherincludes a photon counter portion 915 b comprising a photon counter 912that counts analog voltage pulses of the analog voltage 985 and providesa digitized assay photon count 970 to the interface 925 of theluminometer controller 905.

Returning to FIG. 9A, luminometer controller 905 can be any suitablecomputer system, such as computer system 1500 as described with respectto FIG. 15 . Luminometer controller 905 can include the interface 925, amode determination module 930, a master calibration module 935, a testcycle calibration module 940, a photon count module 945, an analog countmodule 950, an output module 955, and a test result module 960.

The mode determination module 930 can determine the mode of the PMT 630.A master calibration mode can be executed to calibrate the PMT 630 uponinitial build. In some embodiments, the master calibration mode can beexecuted at other times including on a periodic basis, such as monthly.Alternatively, the master calibration mode can be executed upondetermination that the PMT 630 has drifted too far from initialcalibration. A test calibration mode can be executed to calibrate thePMT 630 between test cycles that perform an assay. Optionally, the testcalibration mode can be executed between each test cycle. Optionally,the calibration mode can be executed periodically, such as every 10minutes, for example or after every 10^(th) test cycle as anotherexample. An assay mode can be executed to calculate an assay result.This is also called a test cycle.

The master calibration module 935 can be used when the PMT 630 is inmaster calibration mode. The gain of PMT 630 can be set to operate in aplateau region, as is known in the art of PMTs. The PMT gain can be setby exposing the PMT 630 to a calibrated light source with a known RLUoutput response. For example a NIST (National Institute of Standards andTechnology) approved and traceable calibrated light source may be used.The calibrated light source may be an external light source that is putin the dark chamber 545 formed by closing the cap 415 of the luminometer400. Alternatively, the calibrated light source may include an externallight source and a light source portion of a labyrinth seal, similar tothe cap portion 540 of the labyrinth seal 530, that similarly forms thedark chamber 545 with the light source portion of the labyrinth seal inlieu of the cap portion 540 of the labyrinth seal 530 (and with the cap415 of the luminometer 400 open). Alternatively, the PMT may be removedfrom the chassis 405 and temporarily replaced with a NIST (NationalInstitute of Standards and Technology) approved and traceable calibratedlight sensor. The NIST light sensor may be used to calibrate the LED620. The gain of the PMT 630 may be adjusted until the RLU outputresponse of the PMT 630 corresponds to the calibrated light source. Forexample, the expected RLU output response to the calibrated light sourcemay be 10 million RLUs, so the gain of the PMT 630 may be adjusted untilthe luminometer 400 provides an RLU output response of 10 million RLUs.Once the PMT gain is set, the master calibration module can calculatethe discreet/analog crossover 1015 (as depicted in FIGS. 10A and 10B) ofthe PMT 630. The discreet/analog crossover 1015 may be represented as anRLU value. The discreet/analog crossover 1015, once identified, can be apredetermined value used to identify whether a linear function or anapparent photon count should be relied upon for generating an output ofthe luminometer 400. The relationship between the digitized assay analogvoltage 965 and the digitized assay photon count 970 is used to derive alinear function 1010, as described in detail below. The discreet/analogcrossover 1015 can be calculated using the PMT gain previously set. ThePMT gain is set to a percentage of a maximum gain for that PMT 630. Forexample, the gain of the PMT 630 may have been set to eight-five percentof the maximum PMT gain. Further, a known ideal discreet/analogcrossover value can be used, for example, a known ideal discreet analogcrossover value can be twelve million five hundred thousand RLUs. Theideal discreet/analog crossover value may be determined based on thecircuitry of the luminometer 400. The known ideal discreet/analogcrossover value and the PMT gain are used to calculate thediscreet/analog crossover 1015 for PMT 630. The percentage of the PMTgain multiplied by the known ideal discreet/analog crossover valuedetermines the discreet/analog crossover 1015. For example, eighty-fivepercent of twelve million five hundred thousand RLUs is ten million sixhundred and twenty-five thousand RLUs, which is the discreet/analogcrossover 1015 in this example. The discreet/analog crossover 1015represents the RLU output response value used to determine whether thedigitized assay analog voltage 965 is used to calculate the RLU outputresponse (if the digitized assay photon count 970 is above thediscreet/analog crossover 1015) or if the digitized assay photon count970 is used is reported as the RLU output response (if the digitizedassay photon count 970 is below the discreet/analog crossover 1015). Asdiscussed with respect to FIGS. 2A-2G, as the light input (opticalpower) increases, the photon count becomes saturated due to theoverlapping signal pulses. Accordingly, PMT 630 has a crossover point atwhich the light input is too high to read discrete photon countsaccurately. The discreet/analog crossover 1015 is used to ensure thatthe RLU output response is accurately reported by ensuring that thediscreet photon count is not used above the discreet/analog crossover1015.

Once the discreet/analog crossover 1015 is calculated, the mastercalibration module 935 can calibrate the LED 620 of calibration unit 460(i.e., internal calibration light source). The external calibrationlight source is removed from the system. A high calibration inputvoltage for the LED 620 can be identified by increasing the voltageapplied to the LED 620 until the RLU output response of the PMT 630 isat the discreet/analog crossover 1015. Using the above example, thevoltage applied to the LED is increased until the RLU output response ofthe PMT 630 is ten million six hundred and twenty-five thousand RLUs.The high calibration input voltage value can be saved for later use in amemory of the luminometer computer system (e.g., the luminometercontroller 905). The lowest calibration point P_(C1) (shown in FIG. 10B)can be identified by determining a low calibration input voltage toapply to the LED 620 such that the analog output response of the PMT 630is discernable (measurable). As described in FIG. 10B, the PMT 630 doesnot register an analog component 965 at very low light levels, which isdepicted in FIG. 10B as the discreet photon count only zone Z_(P). Thelow calibration input voltage is identified by lowering the voltageapplied to the LED 620 to zero volts. The voltage can be increasedslowly until an analog output response 965 is detected from the PMT 630.Optionally, an additional buffer voltage (e.g., ten percent) can beapplied to the low calibration input voltage to ensure that the voltageapplied to the LED 620 will result in an analog output response 965 ofthe PMT 630. The RLU output response of the PMT 630 when the lowcalibration input voltage is applied to the LED 620 is depicted in FIG.10B as P_(C1). The low calibration input voltage and the associated RLUoutput response can be saved in a memory of the luminometer computersystem 905.

The master calibration module 935 can generate a linear functionrepresenting curve 1010 as shown in FIGS. 10A and 10B. Curve 1010represents the linear output response of the PMT 630 at any givenoptical power (lower than an overrange zone Z_(O)). The linear functioncan be calculated by first identifying additional calibration points(e.g., P_(C2) and P_(C3)) within a calibration zone Z_(C). The voltagesbetween the low calibration input voltage and the high calibration inputvoltage can be divided into, for example, three segments. The number ofsegments can be more or fewer than three, and the segments canoptionally be equally spaced. In the example of three equal segments,the high calibration input voltage and the low calibration input voltagecan be used as the voltages at the outer bounds of calibration zoneZ_(C), outside points P_(C1) and P_(C4), and two additional voltagevalues, P_(C2) and P_(C3), can be obtained to designate the three equalsegments. As an example, if the low calibration input voltage value istwo volts and the high calibration input voltage value is eight volts,the two intermediate voltage values are four volts and six volts to getthree equal segments. The four calibration input voltage values can eachbe applied to the LED 620 to generate four associated RLU outputresponses from the PMT 630. The four RLU output responses can be used ina linear regression to obtain the linear function slope and offset oflinear curve 1010. The offset value will be an RLU value. The linearfunction (y=m*x+b), using the slope (m) and offset (b), identified usingthe linear regression, is stored in the memory of the luminometercomputer system 905. When graphed, the linear equation is represented bycurve 1010. Stated differently, a light source 620 measured by PMT 630adjusted through the range of optical power from zero to the upperportion of the analog measurement zone Z_(A) will generate the PMToutput response curve 1010. Alternatively, a light source 620 measuredby PMT 630 adjusted through the range of optical power from the lowerportion of the calibration zone Z_(C) to the upper portion of the analogmeasurement zone Z_(A) will generate the PMT output response curve 1010.

FIG. 10A illustrates graph 1000 depicting the analog and discreet RLUoutput response of PMT 630 in relation to the optical power of theinput, according to an embodiment. The curve 1005 represents theapparent photon count 970 as counted by the photon counter 912. As theoptical power increases, the RLU output response, depicted by curve1005, becomes inaccurate because the photon count saturates such thatindividual photons increasingly cannot be counted. The curve 1010represents the RLU output response of PMT 630 based on the digitizedassay analog voltage 965 at optical powers resulting in an RLU outputresponse greater than the discreet/analog crossover 1015 and based onthe digitized apparent assay photon count 970 at optical powers at orbelow the discreet/analog crossover 1015. Curve 1010 further representsthe line extrapolated by master calibration module 935 of FIG. 9A.Because of signal saturation, when the prior luminometer systems andmethods are used to calculate the RLU output of a PMT, the non-linearcurve 1005 is generated. As depicted in the graph 1000, the linearly ofthe RLU output response decreases as the optical power increases whenusing curve 1005. However, testing with known values shows that thedigitized assay analog voltage 965 is accurate for determining the RLUoutput response of the luminometer 400 at optical powers beyond thosethat result in the non-linear RLU output response depicted by curve 1005of FIGS. 10A and 10B and/or curve 305 of FIG. 3 .

Graph 1000 can be used to show the various zones of operability of theluminometer 400. As shown in FIG. 10A, within analog measurement zoneZ_(A), an analog measurement 965 from the conversion circuit 915 can beused to determine the RLU output response of the luminometer 400. Theanalog measurement zone Z_(A) has a lower bound at the discreet/analogcrossover 1015. The discreet/analog crossover 1015 is approximately atthe optical power value (i.e., light power value) of nine picoWatts persquare meter in this example and results in an RLU response ofapproximately eleven million RLUs. The upper bound of the analogmeasurement zone Z_(A) is approximately at optical input of two hundredand forty picoWatts per square meter and approximately an RLU outputresponse of the luminometer 400 of approximately two hundred andninety-five million RLUs. Curve 1010 indicates the accurate and linearRLU output response within the analog measurement zone Z_(A). Asindicated by curve 1005, the RLU output response based on the digitizedapparent assay photon count 970 becomes non-linear within the analogmeasurement zone Z_(A).

The overrange zone Z_(O) falls beyond the analog measurement zone Z_(A).The overrange zone Z_(O) is approximately when the optical power exceedsapproximately two hundred and forty picoWatts per square meter in thisexample. In overrange zone Z_(O), the circuit board and firmware arelimiting. Within overrange zone Z_(O), the optical power exceeds theability of the luminometer 400 to measure and output a valid RLU outputresponse.

As discussed above, the linear portion of the digitized apparent assayphoton count 970 provides an accurate measurement of photons. Thediscreet photon count measurement zone Z_(D) is the zone within whichthe photon counter portion 915 b can identify discreet photons strikingthe photocathode of the PMT 630 and output the photon count. Discreetphoton count measurement zone Z_(D) is within optical power of zero andapproximately nine picoWatts per square meter, in this example. Theupper bound of the discreet photon count measurement zone Z_(D) is thediscreet/analog crossover 1015. The lower threshold of the discreetphoton count measurement zone Z_(D) is zero. The RLU output responsewithin the discreet photon count measurement zone Z_(D) is approximatelybetween zero RLU and eleven million RLU. Within the discreet photoncount measurement zone Z_(D), the indicated RLU output response based onthe apparent digitized assay photon count 970 is linear.

The photon count of the photon counter portion 915 b begins saturatingin the saturation zone Z_(S), when optical power exceeds the discreetphoton count measurement zone Z_(D). Within the saturation zone Z_(S),indicated output from the photon counter 912 of the photon counterportion 915 b as the digitized apparent assay photon count 970 is alower RLU value than the actual RLU value, as indicated by curve 1005.However, within the saturation zone Z_(S), the curve 1010 representingthe RLU output response based on the digitized assay analog voltage 965is accurate and linear. Within the saturation zone Z_(S), the linearfunction generated from the master calibration module 935 is used toobtain the RLU output response based on the digitized assay analogvoltage 965.

FIG. 10B illustrates an enlarged partial view of graph 1000. As shown inFIG. 10B, the RLU output response based on the digitized apparent assayphoton count (shown by curve 1005) indicates that pulse pair resolutionbecomes significant at approximately the discreet/analog crossover 1015.Further, the pulse pair resolution results in saturation atapproximately an RLU output response of one hundred million photons persecond. Calibration point P_(C1), calibration point P_(C2), calibrationpoint P_(C3), and calibration point P_(C4) (e.g., at discreet/analogcrossover 1015) are illustrated, and are shown within the discreetphoton count measurement zone Z_(D) and within the calibration zoneZ_(C). The uppermost calibration point P_(C4) is illustrated as thediscreet/analog crossover 1015 value. The lowest calibration pointP_(C1) is illustrated as the lowest value from which an analog outputresponse can be discerned from the PMT 630 by the analog circuit portion915 a. At optical powers below the lowest calibration point P_(C1) isthe photon counting only zone Z_(P). Within the photon counting onlyzone Z_(P), the conversion circuit 915 of the luminometer 400 outputs aphoton count 970 only (i.e., no analog output 965 is discernable) andphoton counting for RLU output response is the digitized assay photoncount 970 from the conversion circuit 915. Example discreet point 1020within photon counting only zone Z_(P) has an RLU output response valueof 4 RLU, which is the apparent photon value counted per second. At thisoptical power, no analog current component is detectable in the PMToutput signal 975. As illustrated, the calibration zone Z_(C) has anupper bound of the discreet/analog crossover 1015 and a lower bound atthe lowest calibration point P_(C1). Within the calibration zone Z_(C),the digitized apparent assay photon count 970 is used to report the RLUoutput response of the luminometer 400. At optical powers above thecalibration zone Z_(C) (i.e., in the analog measurement zone Z_(A) butnot the overrange zone Z_(O)), the digitized assay analog voltage 970 isused to determine the RLU output response of the luminometer 400. Withinthe calibration zone Z_(C), calibration point P_(C1), calibration pointP_(C2), calibration point P_(C3), and calibration point P_(C4) areobtained to generate the linear function 1010 for use in the analogmeasurement zone Z_(A).

Returning to FIG. 9A, test cycle calibration module 940 can execute atest cycle calibration when the mode determination module 930 determinesthat the luminometer 400 is in test cycle calibration mode. The testcycle calibration module 940 can be used to correct for noise and/ordrift. The calibration can re-linearize the luminometer 400 byrecalculating the linear function. This type of re-linearization may beperformed, for example, once each month. The re-linearization caninclude adjusting the gain of the PMT 630 until the RLU output responseis the discreet/analog crossover 1015 when the high calibration inputvoltage is applied to LED 620. This PMT gain adjustment may help resolvedrift of the PMT 630 and/or account for noise within the system. Afterthe PMT gain is adjusted, the low calibration input voltage is appliedto the LED 620 and the associate RLU output response is saved. The twoadditional calibration input voltages previously determined by themaster calibration module 935 can be applied to the LED 620 and theassociated RLU output response can be obtained at each calibration inputvoltage. The linear regression can be applied to the RLU output responsevalues to obtain the new slope (m) and offset (b) for the linearfunction. The new linear function can then replace the old linearfunction (obtained from the master calibration module 935) and be storedfor use when performing assays.

Mode determination module 930 can determine when an assay is beingperformed. If in assay test mode, the mode determination module 930 candetermine whether the digitized assay photon count 970 is above thediscreet/analog crossover 1015. If the digitized assay photon count 970is above the discreet/analog crossover 1015, the mode determinationmodule 930 can instruct the analog count module 950 to determine the RLUoutput response of the luminometer 400 using the digitized assay analogvoltage 965. If the digitized assay photon count 970 does not exceed thediscreet/analog crossover 1015, the mode determination module 930 caninstruct the photon count module 945 to determine the RLU outputresponse of the luminometer 400 using the digitized assay photon count970.

The photon count module 945 can determine the RLU output response usingthe digitized assay photon count 970 by, for example, reporting thedigitized assay photon count 970.

The analog count module 950 can determine the RLU output response byentering the digitized assay analog voltage 965 into the linear functionthat is saved and was generated by the master calibration module 935 orupdated by the test cycle calibration module 940. The digitized assayanalog voltage 965 can be used as the “x” variable (independentvariable) of the linear function. The slope (m) and offset (b) can beused in the formula RLU output response=y=m*x+b. The analog count module950 can report the RLU output response (i.e., “y” from y=m*x+b).

Each of the master calibration module 935, test cycle calibration module940, photon count module 945, and analog count module 950 can outputinformation, such as an RLU output response for the light detected byPMT 630 to output module 955. Output module 955 can provide informationto a display device 920 for a user to view. Optionally, output module955 can provide the information to test result module 960 for inclusionin a report, for example, to be provided to the patient that providedthe patient sample tested in the assay.

FIG. 11 illustrates a master calibration method 1100 for extendingsignal linearity. The method 1100 can be performed by, for example,luminometer electrical system 900 of FIG. 9A, and more specifically bymaster calibration module 935. The method 1100 can begin with setting again of a light detector 630 using photons emitted from a calibratedlight source at step 1105. As previously discussed, an external lightsource may be introduced into the reaction vessel chamber 610 ofluminometer 400. The RLU output response for the calibrated light sourcemay be known, and a gain of the PMT 630 can be adjusted until the knownRLU output (expected output) response is received.

At step 1110 the master calibration module 935 can calculate thediscreet/analog crossover value 1015 of the light detector (i.e., PMT630) based on the gain. For example, the PMT gain may have been adjustedto a percentage of the maximum gain for the PMT 630. The percentagevalue can be used to identify the discreet/analog crossover value 1015by taking the percentage of an ideal RLU output response. As an example,the ideal RLU output response may be twelve million five hundredthousand RLU. If the PMT gain is set to ninety percent of maximum, forexample, the analog/discreet crossover 1015 is eleven million twohundred fifty thousand RLU.

At step 1115, the master calibration module 935 can calibrate theinternal light source 620 using the discreet/analog crossover value1015. Light source calibration method 1200 is described in FIG. 12 .

FIG. 12 illustrates a light source calibration method 1200, whichprovides additional detail of step 1115 of the master calibration method1100. At step 1205 the master calibration module 935 can determine andsave a high calibration input voltage. The high calibration inputvoltage is determined by increasing the voltage applied to the internallight source (i.e., LED 620) until the RLU output response of theluminometer 400 is the discreet/analog crossover value 1015.

At step 1210 the master calibration module 935 can determine and savethe low calibration input voltage. The low calibration input voltage isdetermined by increasing the voltage applied to the internal lightsource 620 from zero volts until an analog output response is detectedfrom the light detector (i.e. PMT 630). Below the low calibration inputvoltage, the RLU output response from the luminometer 400 is thedigitized assay photon count 970 because there is no digitized assayanalog voltage 965 detected.

At step 1215, the master calibration module 935 can select at least twoadditional calibration input voltages between the high calibration inputvoltage and the low calibration input voltage. For example, two equallydistant input voltages can be calculated and used.

At step 1220, the master calibration module 935 can apply eachcalibration input voltage to the internal light source 620 and store thecalibration input voltage and associated output response of the lightdetector 630. Using four calibration points will result in fourassociated RLU output response values. Any number greater than twocalibration points can be used.

Returning to FIG. 11 , at step 1120, the master calibration module 935can apply a linear regression to the RLU output response values obtainedfrom applying the calibration input values to the LED 620 to generate alinear function slope (m) and offset (b). The linear function representsthe extended linear response of the PMT (light detector) for RLU valuesup to 20 million RLUs. Above 20 million RLUs, photoelectron pulses startto combine causing the curve 1005 to bend and thus become non-linear.

At step 1125, a sample may be run. FIG. 13 illustrates a flowchartdepicting a method 1300 for performing an assay 100 with luminometer400. Method 1300 begins at step 1305 with placing a light emittingsample in a reaction vessel 105. A patient sample may first be placed ina reaction vessel 105, followed by various reagents and/or substratematerials. The light emitting sample may be generated through theprocess described in assay 100 with respect to FIG. 1 that results inthe light emitting sample at stage 8. The reaction vessel (e.g.,reaction vessel 105) can be placed in a reaction vessel chamber (e.g.,reaction vessel chamber 610). At step 1310, a PMT (e.g., PMT 630) canmeasure photons emitted from the processed sample for a predeterminedperiod of time (e.g., 1 second). The output signal 975 from the PMT 630can be captured incrementally over the period of time and averaged(using boxcar averaging) to generate a digitized apparent assay photoncount 970 and a digitized assay analog voltage 965 by, for example,conversion circuit 915 as shown at optional step 1345. For example, theluminometer 400 may examine the light output of one processed sampleevery instrument pitch (e.g., 8 seconds). Within the instrument pitch,the luminometer 400 may perform three separate reads of the sample. Eachof the reads may be performed over a one second time period. The photoncounter portion 915 b thereby counts apparent photons from the processedsample three times while the processed sample is within the dark chamber545 and derives a count for each of the three times. The reporteddigitized assay photon count 970 may be the middle (i.e., median) valueof the three counts. By reporting the median value as the digitizedassay photon count 970, outlier results, which may be caused by cosmicrays, X-rays, and/or other effects not from the sample, are rejected.The analog circuit portion 915 a also collects one result over each ofthe three separate reads of the sample. In particular, during each onesecond sample read, 10,000 measurements of the voltage values 980 areaveraged and reported as the digitized assay analog voltage 965 as shownat optional step 1345. However, only one of these three averaged valuesis used as the output further processed by the luminometer 400. Forexample, the averaged value that corresponds to the time period of themedian value of the digitized assay photon count 970 may be used. Theluminometer computer system (e.g., luminometer controller 905) canreceive the digitized assay photon count 970 and digitized assay analogvoltage 965 and determine, at step 1320, whether the digitized assayphoton count 970 exceed the discreet/analog crossover 1015. If thedigitized assay photon count 970 exceeds the discreet/analog crossover1015, the method 1300 continues at step 1325 to calculate the assayanalog voltage 965 using the assay analog current value. Optionally, theassay analog voltage 965 can be calculated before determining whetherthe digitized assay photon count 970 exceeds the discreet/analogcrossover 1015. At step 1330 the luminometer controller 905 cancalculate the RLU output response using the digitized assay analogvoltage 965 and the linear function. At step 1335, the luminometercontroller 905 can report the RLU output response. If the digitizedassay photon count 970 did not exceed the discreet/analog crossover 1015at step 1320, the luminometer controller 905 can report the digitizedapparent assay photon count 970 as the RLU output response at step 1340.

FIG. 14 illustrates a flowchart depicting a calibration method 1400. Themethod 1400 can be performed by, for example luminometer electricalsystem 900 of FIG. 9A. At step 1405, the test cycle calibration module940 can apply the high calibration input voltage to the internal lightsource (i.e., LED 620) and adjust the gain of the light detector (i.e.,PMT 630) to obtain an RLU output response that equals thediscreet/analog crossover 1015. The RLU output response is obtainedusing the digitized assay photon count 970. At step 1410 the test cyclecalibration module 940 can apply each calibration input voltage to theinternal light source and store the associated RLU output response ofthe light detector 630. The associated RLU output response for thecalibration in the calibration zone Z_(C) may be the digitized assayphoton count 970. The calibration input voltages were each determinedduring the master calibration as described in method 1200. At step 1415,the test cycle calibration module 940 can apply a second linearregression to the most recent RLU output response values obtained usingthe calibration input voltages. The linear regression will provide anupdated slope (m) and offset (b) for the linear function. At step 1420,the test cycle calibration module 940 can replace the old linearfunction with the new linear function in the luminometer computer systemmemory.

FIG. 15 illustrates a block diagram of an example computer system 1500usable for performing signal linearity extension as described in detailherein. The computing device 1500 can be or include, for example, alaptop computer, desktop computer, tablet, e-reader, smart phone ormobile device, smart watch, personal data assistant (PDA), or otherelectronic device.

The computing device 1500 can include a processor 1640 interfaced withother hardware via a bus 1505. A memory 1510, which can include anysuitable tangible (and non-transitory) computer readable medium, such asRAM, ROM, EEPROM, or the like, can embody program components (e.g.,instructions 1515) that configure operation of the computing device1700. In some examples, the computing device 1500 can includeinput/output (“I/O”) interface components 1525 (e.g., for interfacingwith a display 1545, keyboard, or mouse) and additional storage 1530.

The computing device 1500 can include network components 1520. Networkcomponents 1520 can represent one or more of any components thatfacilitate a network connection. In some examples, the networkcomponents 1520 can facilitate a wireless connection and includewireless interfaces such as IEEE 802.11, Bluetooth, or radio interfacesfor accessing cellular telephone networks (e.g., a transceiver/antennafor accessing CDMA, GSM, UMTS, or other mobile communications network).In other examples, the network components 1520 can be wired and caninclude interfaces such as Ethernet, USB, or IEEE 1394.

Although FIG. 15 depicts a single computing device 1500 with a singleprocessor 1540, the system can include any number of computing devices1500 and any number of processors 1540. For example, multiple computingdevices 1500 or multiple processors 1540 can be distributed over a wiredor wireless network (e.g., a Wide Area Network, Local Area Network, orthe Internet). The multiple computing devices 1500 or multipleprocessors 1540 can perform any of the steps of the present disclosureindividually or in coordination with one another.

Each of the calculations or operations described herein may be performedusing a computer or other processor having hardware, software, and/orfirmware. The various method steps may be performed by modules, and themodules may comprise any of a wide variety of digital and/or analog dataprocessing hardware and/or software arranged to perform the method stepsdescribed herein. The modules optionally comprising data processinghardware adapted to perform one or more of these steps by havingappropriate machine programming code associated therewith, the modulesfor two or more steps (or portions of two or more steps) beingintegrated into a single processor board or separated into differentprocessor boards in any of a wide variety of integrated and/ordistributed processing architectures. These methods and systems willoften employ a tangible media embodying machine-readable code withinstructions for performing the method steps described above. Suitabletangible media may comprise a memory (including a volatile memory and/ora non-volatile memory), a storage media (such as a magnetic recording ona floppy disk, a hard disk, a tape, or the like; on an optical memorysuch as a CD, a CD-R/W, a CD-ROM, a DVD, or the like; or any otherdigital or analog storage media), or the like.

Different arrangements of the components depicted in the drawings ordescribed above, as well as components and steps not shown or describedare possible. Similarly, some features and sub-combinations are usefuland may be employed without reference to other features andsub-combinations. Embodiments of the present disclosure have beendescribed for illustrative and not restrictive purposes, and alternativeembodiments will become apparent to readers of this patent. In certaincases, method steps or operations may be performed or executed indiffering order, or operations may be added, deleted or modified. It canbe appreciated that, in certain aspects of the present disclosure, asingle component may be replaced by multiple components, and multiplecomponents may be replaced by a single component, to provide an elementor structure or to perform a given function or functions. Except wheresuch substitution would not be operative to practice certain embodimentsof the present disclosure, such substitution is considered within thescope of the present disclosure.

It is to be understood that the figures and descriptions of embodimentsof the present disclosure have been simplified to illustrate elementsthat are relevant for a clear understanding of the present disclosure.Those of ordinary skill in the art will recognize, however, that theseand other elements may be desirable. However, because such elements arewell known in the art, and because they do not facilitate a betterunderstanding of the present disclosure, a discussion of such elementsis not provided herein. It should be appreciated that the figures arepresented for illustrative purposes and not as construction drawings.Omitted details and modifications or alternative embodiments are withinthe purview of persons of ordinary skill in the art.

The examples presented herein are intended to illustrate potential andspecific implementations of the present disclosure. It can beappreciated that the examples are intended primarily for purposes ofillustration of the present disclosure for those skilled in the art.There may be variations to these diagrams or the operations describedherein without departing from the spirit of the present disclosure.

Furthermore, whereas particular embodiments of the present disclosurehave been described herein for the purpose of illustrating the presentdisclosure and not for the purpose of limiting the same, it will beappreciated by those of ordinary skill in the art that numerousvariations of the details, materials and arrangement of elements, steps,structures, and/or parts may be made within the principle and scope ofthe present disclosure without departing from the present disclosure asdescribed in the claims.

All patents, patent publications, patent applications, journal articles,books, technical references, and the like discussed in the instantdisclosure are incorporated herein by reference in their entirety forall purposes.

What is claimed is: 1.-19. (canceled)
 20. A luminometer comprising: alight detector configured to: receive photons emitted from a lightsource; and output a detector signal indicating a strength of the lightsource based on a number of photons received, wherein the detectorsignal comprises an analog component and a discreet component; aconversion circuit configured to: receive the detector signal from thelight detector; generate a digitized signal from the analog component ofthe detector signal, the digitized signal representing the strength ofthe light source; generate a digitized photon count from the discreetcomponent of the detector signal, the digitized photon countrepresenting an apparent number of photons received by the lightdetector from the light source; and output the digitized signal and thedigitized photon count; a luminometer controller; and aprocessor-readable memory having stored therein a set of instructionswhich, when executed by the luminometer controller, cause theluminometer controller to: receive the digitized photon count; comparethe digitized photon count to a discreet-analog crossover value; outputthe digitized photon count as a result from the luminometer when thedigitized photon count does not exceed the discreet-analog crossovervalue; and when the digitized photon count exceeds the discreet-analogcrossover value: apply a calibration function to the digitized signal tocalculate an output value; and output the output value as the resultfrom the luminometer.
 21. The luminometer of claim 20, wherein theluminometer controller is further configured to: perform a mastercalibration in which a photon measurement processor: sets a gain of thelight detector to operate in a plateau region; calculates thepredetermined value using the gain of the light detector; calculates alow voltage value of the light detector using the gain of the lightdetector; and generates the linear function using the predeterminedvalue and the low voltage value.
 22. The luminometer of claim 21,further comprising: a light emitting diode arranged such that the lightdetector measures photons emitted by the light emitting diode; andwherein the luminometer controller is configured to: apply a minimalvoltage to the light emitting diode; increase the minimal voltage to aminimum readable voltage, the minimum readable voltage being a minimumvoltage applied to the light emitting diode to cause the light emittingdiode to emit sufficient photons for the light detector to measure;receive an LED photon count and an LED analog signal value eachrepresenting the photons emitted from the light emitting diode when theminimum readable voltage is applied to the light emitting diode; andsave the LED analog signal value as the low voltage value.
 23. Theluminometer of claim 21, further comprising: a light emitting diodearranged such that the light detector measures photons emitted from thelight emitting diode; a light sensor configured to sense light output ofthe light emitting diode and to generate an LED analog output signalvalue representing photons emitted from the light emitting diode; andwherein the luminometer controller is configured to: apply a minimumreadable voltage to the light emitting diode; increase the minimumreadable voltage by a buffer voltage until the LED analog output signalvalue representing the photons emitted from the light emitting diodewhen the minimum readable voltage plus the buffer voltage is applied tothe light emitting diode is greater than the low voltage value plus abuffer voltage value, wherein the buffer voltage value represents thephotons emitted from the light emitting diode when the buffer voltage isapplied to the light emitting diode; and save the LED analog outputsignal value as the predetermined value.
 24. The luminometer of claim21, further comprising: a light emitting diode arranged such that thelight detector measures photons emitted from the light emitting diode;and wherein the luminometer controller is configured to: determine aminimum readable signal that generates the low voltage value from thelight detector when the minimum readable signal is applied to the lightemitting diode; determine a high signal that generates the predeterminedvalue from the light detector when the minimum readable signal plus abuffer voltage is applied to the light emitting diode; select a firstvoltage and a second voltage between the high voltage and the minimumreadable voltage that divides the minimum readable voltage to the highvoltage into four equal increments; receive a plurality of calibrationanalog signal values and a plurality of calibration photon counts fromthe light detector based on applying each of the minimum readablevoltage, the high voltage, the first voltage and the second voltage tothe light emitting diode; and perform a linear regression using theplurality of calibration analog signal values and the plurality ofcalibration photon counts to generate the linear function.
 25. Theluminometer of claim 20, wherein the luminometer controller isconfigured to: calculate the photon count by accumulating pulse countsfrom the light detector over the predetermined period of time using aboxcar averaging function.
 26. The luminometer of claim 20, wherein theluminometer controller is configured to: convert and filter an outputsignal of the light detector to generate an analog output voltage;collect the analog output voltage in increments over the predeterminedperiod of time to generate a plurality of filtered voltage increments;and average the filtered voltage increments to generate the analogvoltage value.
 27. The luminometer of claim 20, further comprising: alight emitting diode arranged such that the light detector measuresphotons emitted from the light emitting diode; and wherein theluminometer controller is configured to perform a test calibration cycleto correct a drift of the light detector, wherein the test cyclecalibration is based on the predetermined value and a low voltage value,and wherein the low voltage value represents photons emitted from thelight emitting diode when a minimum readable voltage is applied to thelight emitting diode.
 28. The luminometer of claim 20, wherein theanalog circuit includes a first amplifier and wherein a signal to noiseratio at an input to the first amplifier is greater than 50 and lessthan 1,000,000.
 29. The luminometer of claim 20, wherein the analogcircuit includes a first amplifier and wherein a signal to noise ratioat an input to the first amplifier is greater than 500 and less than4,000.
 30. The luminometer of claim 20, wherein a signal to noise ratioat an input to the analog circuit is greater than
 100. 31. Theluminometer of claim 20, wherein the analog signal is an analog voltage.32. The luminometer of claim 20, wherein the light detector includes aphotomultiplier tube.
 33. The luminometer of claim 20, wherein: thelight detector is a photomultiplier tube configured to sense photonsemitted from an assay reaction over a period of time; the conversioncircuit includes an analog circuit configured to provide an assay analogvoltage based on the photons emitted from the assay over the period oftime, the analog circuit comprising: a current sensing resistor coupledto convert current from the photomultiplier tube to a voltage; anamplifier configured to amplify the voltage; and a dedicated electricalconnection between a terminal of the current sensing resistor and aterminal of the amplifier; the conversion circuit further includes acounter circuit configured to provide an assay photon count based on thephotons emitted from the assay over the period of time; and theluminometer controller is configured to: in response to the assay analogvoltage being greater than a predetermined value, calculate a relativelight unit value of the photons emitted from the assay over the periodof time based on the assay analog voltage and an optimized linearfunction.
 34. The luminometer of claim 33, wherein; the current sensingresistor and the amplifier are disposed on a printed circuit boardhaving a common ground plane; and the terminal of the current sensingresistor and the terminal of the amplifier are connected to the commonground plane.